M. Balasch

Experimental Inoculation of Conventional Pigs with Porcine Reproductive and Respiratory Syndrome Virus and Porcine Circovirus 2

ABSTRACT Postweaning multisystemic wasting syndrome (PMWS) is a disease of nursery and fattening pigs characterized by growth retardation, paleness of the skin, dyspnea, and increased mortality rates. Porcine circovirus 2 (PCV2) has been demonstrated to be the cause of PMWS. However, other factors are needed for full development of the syndrome, and porcine reproductive and respiratory syndrome virus (PRRSV) infection has been suggested to be one of them. Twenty-four conventional 5-week-old pigs were distributed in four groups: control (n = 5), PRRSV inoculated (n = 5), PCV2 inoculated (n = 7), and PRRSV and PCV2 inoculated (n = 7). The two groups inoculated with PRRSV showed growth retardation. Pigs inoculated with both PRRSV and PCV2 had increased rectal temperature. One of these pigs developed wasting, had severe respiratory distress, and died. The most important microscopic lesion in pigs inoculated with PCV2 was lymphocyte depletion with histiocytic infiltration of the lymphoid organs, more severe and in a wider range of tissues in doubly inoculated pigs. Interstitial pneumonia was observed in the three inoculated groups. PCV2 nucleic acid was found by in situ hybridization in larger amounts and in a wider range of lymphoid tissues in PRRSV- and PCV2-inoculated than in PCV2-inoculated pigs. TaqMan PCR was performed to quantify the PCV2 loads in serum during the experiment. PCV2 loads were higher in doubly inoculated pigs than in pigs inoculated with PCV2 alone. These findings indicate that severe disease can be reproduced in conventional 5-week-old pigs by inoculation of PRRSV and PCV2. Moreover, these results support the hypothesis that PRRSV infection enhances PCV2 replication.

Identification of porcine circovirus in tissues of pigs with porcine dermatitis and nephropathy syndrome

Thirty-three pigs affected by porcine dermatitis and nephropathy syndrome, 30 from Spain and three from the USA, were investigated in order to detect porcine circovirus (Pcv) in their tissues. A standard in situ hybridisation technique using a specific DNA 317-bp probe based on a well-conserved sequence of Pcv (which recognises both Pcv-i and PCV-2) was applied to formalin-fixed, paraffin-embedded tissues. Twentyeight of the 30 Spanish pigs and all three American pigs had Pcv in at least one tissue. Viral nucleic acid was detected mainly in lymphoid organs, and especially the lymph nodes. The viral genome was also found, in order of decreasing quantity, in Peyers patches, tonsil, lung, spleen, kidney, liver, and skin. Viral nucleic acid was located mainly within the cytoplasm of monocyte/macrophage lineage cells, including follicular dendritic cells, macrophages, histiocytes and Kupffer cells. No viral nucleic acid was found in damaged glomeruli or arteriolar walls. In frozen samples available from three Spanish pigs, the virus was identified as type 2 by using the polymerase chain reaction and restriction fragment length polymorphism. Most of the pigs from which serum was available were seropositive against porcine respiratory and reproductive syndrome virus (PRRSV), and PRRSV antigen was detected in the lung of two of the Spanish pigs. These results suggested that Pcv is present in tissues of almost all pigs affected by PDNS, and Pcv has to be considered as a possible agent involved in the pathogenesis of the syndrome.

Serum antibodies to porcine circovirus type 1 and type 2 in pigs with and without PMWS

PORCINE circovirus (PCV) is a small, single-stranded, circular, covalently-closed DNA virus, which was first detected as a contaminant in the pig kidney cell line PK-15 (ATCC CCL-33) (Tischer and others 1974). PK-15-derived PCV is considered apathogenic (Tischer and others 1986, Allan and others 1995), and serological studies for the detection of PCV antibodies in pig sera from Germany, Canada, New Zealand, Great Britain, Northern Ireland and the USA have shown a widespread presence of PCV antibodies in fattening and adult pigs, ranging from 25 to 98 per cent of the investigated sera. Therefore, it has been suggested that PCV infection is ubiquitous throughout the world (Allan 1996). Since 1991 in Canada (Clark 1997) and during the past two years in several other countries, including the USA, France, Spain, the UK, Denmark, Italy and Germany, a new disease called postweaning multisystemic wasting syndrome (PMWS) has been identified. PMWS affects nursery and fattening pigs, and i s histologically characterised by lymphocyte depletion with histiocytic infiltrates within lymphoid organs, and bronchointerstitial pneumonia (Clark 1997, Segalés and others 1997). An unequivocal aetiological relationship between PCV and PMWS has not been demonstrated; however, pigs affected with PMWS are regularly infected with a novel strain of PCV. This new strain of PCV has a nucleotide sequence similarity of 75 per cent when compared with PCV derived from PK-15 cells (Hamel and others 1998, Meehan and others 1998); therefore, the nomenclature PCV type 1 (PCV-1) and PCV type 2 (PCV-2) has been suggested for the PK-15and PMWS-derived PCV, respectively (Allan and others 1999). Antigenic differences between PCV-1 and PCV-2 do exist, as some monoclonal antibodies raised against PCV-1 are not able to recognise PCV-2, and monoclonal antibodies raised against PCV-2 cannot recognise PCV-1 in infected cell monolayers by immunofluorescence (Allan and others 1999). In addition, hyperimmune rabbit serum raised by immunisation with PCV-2 showed a high specific titre against PCV-2 but only a low titre against PCV-1 in cell monolayers (Allan and others 1998). The aim of this study was to compare serum antibody titres against PCV-1 and PCV-2 in necropsied pigs, with and without PMWS, using an immunoperoxidase monolayer assay (IPMA) technique. A total of 90 oneto five-month-old pigs from 37 different Spanish farms were submitted to the laboratory of the Veterinary School of Barcelona for pathological examination between May 1997 (when PMWS was recognised in Spain) and July 1998. Pigs were bled at the jugular vein and a 10 ml blood sample was collected in a Vacutainer (Venoject; Terumo Europe). The sample was allowed to clot, centrifuged and then the serum was frozen at –80°C until required for testing. The pigs were necropsied and tissue samples (including the lungs, lymphoid organs, livers and kidneys) were fixed in 10 per cent buffered formalin, embedded in paraffin wax and routinely processed for histopathology. Pigs were classified as PCV-2Short Communications

Transmissible gastroenteritis coronavirus gene 7 is not essential but influences in vivo virus replication and virulence

Abstract Transmissible gastroenteritis coronavirus (TGEV) contains eight overlapping genes that are expressed from a 3′-coterminal nested set of leader-containing mRNAs. To facilitate the genetic manipulation of the viral genome, genes were separated by duplication of transcription regulating sequences (TRSs) and introduction of unique restriction endonuclease sites at the 5′ end of each gene using an infectious cDNA clone. The recombinant TGEV (rTGEV) replicated in cell culture with similar efficiency to the wild-type virus and stably maintained the modifications introduced into the genome. In contrast, the rTGEV replication level in the lungs and gut of infected piglets and virulence were significantly reduced. rTGEV in which gene 7 expression was abrogated (rTGEV-Δ7) were recovered from cDNA constructs, indicating that TGEV gene 7 was a nonessential gene for virus replication. Interestingly, in vivo infections with rTGEV-Δ7 showed an additional reduction in virus replication in the lung and gut, and in virulence, indicating that TGEV gene 7 influences virus pathogenesis.

Engineering the transmissible gastroenteritis virus genome as an expression vector inducing lactogenic immunity.

ABSTRACT The genome of the coronavirus transmissible gastroenteritis virus (TGEV) has been engineered as an expression vector with an infectious cDNA. The vector led to the efficient (>40 μg/106 cells) and stable (>20 passages) expression of a heterologous gene (green fluorescent protein [GFP]), driven by the transcription-regulating sequences (TRS) of open reading frame (ORF) 3a inserted in the site previously occupied by the nonessential ORFs 3a and 3b. Expression levels driven by this TRS were higher than those of an expression cassette under the control of regulating sequences engineered with the N gene TRS. The recombinant TGEV including the GFP gene was still enteropathogenic, albeit with a 10- to 102-fold reduction in enteric tissue growth. Interestingly, a specific lactogenic immune response against the heterologous protein has been elicited in sows and their progeny. The engineering of an additional insertion site for the heterologous gene between viral genes N and 7 led to instability and to a new genetic organization of the 3′ end of the recombinant viruses. As a consequence, a major species of subgenomic mRNA was generated from a TRS with the noncanonical core sequence 5′-CUAAAA-3′. Extension of the complementarity between the TRS and sequences at the 3′ end of the viral leader was associated with transcriptional activation of noncanonical core sequences. The engineered vector led to expression levels as high as those of well-established vectors and seems very promising for the development of vaccines and, possibly, for gene therapy.

A genetically engineered chimeric vaccine against porcine circovirus type 2 (PCV2) improves clinical, pathological and virological outcomes in postweaning multisystemic wasting syndrome affected farms

The present study describes the effects of a commercially available genetically engineered chimeric vaccine against porcine circovirus type 2 (PCV2) on clinical, pathological and virological features in three multi-site farms suffering from postweaning multisystemic wasting syndrome (PMWS). The vaccine product was able to reduce clinical signs, PCV2 viral load in lymphoid organs and/or sera, and overall mortality in nurseries and fattening units. This is the first time in which is shown that a PCV2 vaccine is able to decrease specifically PMWS-associated mortality. Another novelty of this study is the assessment of PMWS-like histological lesions in a large number of vaccinated and non-vaccinated pigs under field conditions.

Transient correlation between viremia levels and IL-10 expression in pigs subclinically infected with porcine circovirus type 2 (PCV2)

Abstract Immunological impairment by porcine circovirus type 2 (PCV2) infection is well documented in pigs suffering from postweaning multisystemic wasting syndrome. Nonetheless, little is known about immune status of pigs that remain PCV2 subclinically infected. Thus, seven pigs successfully infected in an experimental inoculation and without developing disease and nine control non-inoculated pigs were examined. Serological, virological and immunological determinations were done throughout ten weeks post-infection (PI). At week 3 PI, inoculated animals presented the peak of viremia and produced higher levels of IL-10 than the controls; correlation between viral load and IL-10 amounts was observed (p <0.05). Also, the ratio IgM/IgG suffered a shift skewing IgM production towards an IgG response. By 10 weeks PI, levels of IL-10 disappeared and the viremia decreased. In summary, subclinically PCV2-infected pigs developed a transient PCV2-specific IL-10 response during the viremic phase of infection which coincided with the inversion of the IgM/IgG ratio.

Genotypic shift of porcine circovirus type 2 from PCV-2a to PCV-2b in Spain from 1985 to 2008

Changes in porcine circovirus type 2 (PCV-2) genotypes were evaluated before, during and after outbreaks of post-weaning multisystemic wasting syndrome (PMWS) in (1) a retrospective study using pig sera collected in Spain from 1985 to 2008 and (2) a longitudinal study using pig sera collected from two farms in Spain over periods of 7 and 14 years. In both studies, there was a rapid genotypic shift from PCV-2a to PCV-2b that was related to the peak of PMWS epizootics in Spain and the appearance of PMWS on the two farms studied longitudinally.

In vitro and in vivo expression of foreign genes by transmissible gastroenteritis coronavirus-derived minigenomes

A helper-dependent expression system based on transmissible gastroenteritis coronavirus (TGEV) has been developed using a minigenome of 3.9 kb (M39). Expression of the reporter gene beta-glucuronidase (GUS) (2-8 microg per 10(6) cells) and the porcine respiratory and reproductive syndrome virus (PRRSV) ORF5 (1-2 microg per 10(6) cells) has been shown using a TGEV-derived minigenome. GUS expression levels increased about eightfold with the m.o.i. and were maintained for more than eight passages in cell culture. Nevertheless, instability of the GUS and ORF5 subgenomic mRNAs was observed from passages five and four, respectively. About a quarter of the cells in culture expressing the helper virus also produced the reporter gene as determined by studying GUS mRNA production by in situ hybridization or immunodetection to visualize the protein synthesized. Expression of GUS was detected in the lungs, but not in the gut, of swine immunized with the virus vector. Around a quarter of lung cells showing replication of the helper virus were also positive for the reporter gene. Interestingly, strong humoral immune responses to both GUS and PRRSV ORF5 were induced in swine with this virus vector. The large cloning capacity and the tissue specificity of the TGEV-derived minigenomes suggest that these virus vectors are very promising for vaccine development.

Study of the persistence of Aujeszky's disease (pseudorabies) virus in peripheral blood mononuclear cells and tissues of experimentally infected pigs

The presence of Aujeszkys disease virus (ADV) in peripheral blood mononuclear cells (PBMC) and tissues of experimentally infected pigs was studied. Vaccinated and unvaccinated pigs were inoculated with different doses of Aujeszkys disease NIA-3 strain. Pigs were periodically bled and PBMC were used for virus isolation and PCR detection of virus. Tissues were obtained at the time of death (8 weeks post-inoculation) and used for ADV genome detection by PCR. ADV genome was amplified from PBMC during the acute phase of infection and, in some experimental groups, up to 38 days post-inoculation (PI). The virus was sporadically detected by virus isolation performed from PBMC. In neural tissues, ADV was constantly amplified from the trigeminal ganglia and the olfactory bulb of persistently infected pigs (euthanized 8 weeks PI). In other tissues, the viral genome was rarely detected in lymph nodes and tonsils, and occasionally, in the bone marrow. Our results indicated that PBMC are not an appropriate source for detecting ADV persistence, since inconsistent results were obtained throughout the experiments. In neural tissues, the olfactory bulb turned out to be as important a target for ADV persistence as the trigeminal ganglia. Viral genome detection in the bone marrow indicated that this tissue may play a role in the establishment of a persistent infection.